Micropropagation proves very effective for the multiplication in conformity, fast and in great number of plants selected for their interesting agronomic properties: rootstocks and varieties of fruit species, forest elite selections, ornamental trees with single flowering or growth behaviour. The fruit- nurseries need virus free plant material to supply the fruit growers. Meristem culture allows viruses eradication and the establishment of healthy mother-plant material. This material must also be preserved from any contamination during biological indexing period. Our work contributes to setting production systems of healthy plant material.
Description of tasks
Viral eradication from fruit species Direct removing of meristems (0.1 mm) from the vegetative buds of branches in post-dormancy stage leads to the eradication of the majority of the viruses which contaminate the cherry, plum or apple trees. Some like Necrotic Ring Spot Virus (NRSV) and the Plum Dwarf Virus (PDV) contaminating Prunus species, however require a second meristem culture from microshoots conditionned in thermotherapy. On the other hand, in active period of tree growth, the genotypes are established starting from nodes; the meristem culture taking then place later on established culture. Production of healthy plant material The method which consists in rooting the mericlones and growing them on their own roots in the field, require sometimes several years. The ex vitro micrografting of microshoots on healthy rootstock issued from in vitro either directly or after a classical multiplication procedure (cutting, mini-layering) ensures the transfer of the mericlones to the greenhouse after the first axillary buds appeared; it reduces the in vitro culture period to a few months instead of 1 to 2 years. Although the method of mass micropropagation of healthy fruit species was developed in our laboratory for a very great number of species and genotypes (more than 500) of various, any new accession represents an opportunity of optimizing the various phases of culture (successively axillary budding, shoot elongation, rooting and acclimatization). It is also about always better controlling certain recurring physiological problems such as hyperhydricity and apex necrosis for example, the formation of the rooting system in or ex vitro and the growth of the plantlets during acclimatization in order to improve quality of the vitroplants. Alternatives to the micropropagation Micropropagation remains an expensive technique and requires setting up by specialized laboratories. It is necessary thus to seek alternative methods of plant multiplication when the economical value of the vitroplants is the limiting factor compared to seeds, cuttings or layering plants. Moreover, the juvenility of the vitroplants supports cuttings propagation procedure. Once mother-plants miniaturized in greenhouse, cuttings are collected the all year long for a polyclonal variety of elites wild cherry trees. The propagation of Camil cherry tree rootstock under these microcuttings conditions reached 1500 plants a year from per square meter. Suckering and layering of M9/Kl 29 apple rootstock is under evaluation in greenhouse. Preservation of mericlones The meristematic lines must be maintained in vitro till the end of the biological indexing in the field. They are currently in slow growth, at +2C °for a period from 3 to 4 years according to the species. A longer-term conservation method must be required to mitigate any putative resurgence or new viral contamination of the mother plants. A cryoconservation technology is considered for longer term preservation of buds. In vitro biological indexing The elimination of the viruses is confirmed only after several years of field indexing. The molecular analyses carried out at the CRA-W are used to detect certain viruses precociously. They do not replace however the biological diagnosis. With the NRSV, the in vitro micrografting of contaminated microshoots causes the necrosis of Shirofugen indicator within the next 10 days.
Expected results
- Precocious in vitro detection of healthy mericlones by biological indexing and molecular tools. - Cryoconservation of mericlones - Characterization of the behaviour along the in vitro procedure
